Protein S-palmitoylation is a dynamic post-translational modification attaching the 16-carbon fatty acid (palmitate) to the cysteine residues. Genetic studies have shown that 23 of DHHC (Asp-His-His-Cys) domain-containing enzymes are protein palmitoyl acyltransferases (PATs). In addition, some proteins could undergo non-enzymatic autopalmitoylation when bound to palmitoyl-Coenzyme A (CoA) directly. To explore their functions, we have developed novel activity-based chemical probes to covalently label and profile PATs and autopalmitoylated proteins in cells. Through proteomic, biochemical and structural studies, we have discovered that the mammalian TEA domain family (TEAD/TEF) transcription factors (TEAD1-4) are autopalmitoylated proteins. TEADs are essential transcription factors that bind to the transcription co-activators YAP/TAZ and regulate Hippo pathway transcriptional output. Deregulation of TEAD-YAP complex has been linked to defective regeneration and organ size control in liver, skin, colon, and heart. Therefore, understanding this novel regulation will shed light on how fatty acid metabolism and autopalmitoylation regulate transcription factors. Our specific aims of this proposal include: (1) To determine the regulatory mechanism(s) of TEADs autopalmitoylation. (2) To investigate the roles of TEAD palmitoylation in regulating Hippo pathway in vitro and in vivo. (3) To develop small molecule inhibitors of TEAD autopalmitoylation as chemical tools.